Research Area: (06) Enabling Technologies Challenge topic: New technologies for neuroscience research (06-MH-103) Project Title: Development of Split-DamID as an alternative methodology to chromatin immunoprecipitation Project Abstract A wide variety of genetic or drug-induced mental health disorders have been linked to altered functions of transcription factors, be it drug-induced or caused by genetic defects. Although it is known that the expression patterns of many genes are altered in the brain after exposure to a psychotropic drug, ultimately we must determine which genes were directly activated by the relevant transcription factors to uncover the expression hierarchy and prioritize druggable targets. Progress towards identification of these "direct targets" has been slow since the traditional methods used to identify the immediate targets of a transcription factor (chromatin IP or ChIP) are expensive, labor-intensive and technically demanding. Most dauntingly, they require very good antibodies and a large amount of starting material, largely limiting their use to cells in culture. In order to provide an approach that avoids these issues, we have optimized the sensitivity and specificity of an alternative technology, DamID, for broad use in neuroscience. Our solution (Split-DamID) relies on our ability to successfully split the bacterial DNA adenine methyltransferase (Dam) into N-terminal (D) and C-terminal (AM) halves. Each half is inactive;however, when fused to interacting transcription factors, the halves will reconstitute enzymatic activity only at the binding sites where these transcription factors interact. The reconstituted Dam marks adenine (A) by addition of a methyl group (Am) in the sequence GAmTC found in proximity to most cognate binding sites of any transcription factor. It is important to note that this epigenetic modification is never found in eukaryotic DNA and appears to have no consequence to cellular or organism survival. This GAmTC mark allows isolation of the transcription factor bound DNA from total DNA through digestion with Dpn1, an enzyme that digests only DNA methylated at GAmTC sites, and adaptor mediated PCR. We have successfully reconstituted Dam activity when fused to Notch or Mef2c. Importantly, we have observed enrichment of known Notch targets over other DNA fragments in cells reconstituting split-Dam fused to Notch. Split-DamID has several key advantages: much improved signal to noise ratio compared with ChIP or DamID;adenine methylation indelibly marks DNA bound by the fusion protein, allowing identification of transient interactions;the recovery of marked DNA is independent of antibody availability;and the amplification of the marked DNA by adaptor mediated PCR makes it amenable to working with the endogenous expression level of the fusion protein and low amounts of starting material. Moreover, Spilt-DamID permits precise spatial and temporal control of Dam reconstitution. We propose to generate multiple Split-Dam pairs with mental health related factors (Notch, DeltaFosB, CREB, NF[unreadable]B, and Mef2) and common transcription co-activators (p300, others) to reconstitute Dam activity only at sites where transcription takes place. These targeting vectors, expression vectors, cells and animals will create a resource for investigators interested in identifying sites directly bound by transcription factors implicated in mental health in animal models exposed to psychotropic drugs or carrying a mutation known to affect behavior in humans. We will develop resources allowing investigators interested in brain function to mark and identify key target genes bound by transcription factors of interest (e.g., those involved in mental health, cancer, and other transcription-driven processes in neuroscience). They will have control over when the marking occur, enabling precise correlation of transcriptional activity and behavior. In contrast to the current technology, our novel method will work for any transcription factor and requires only a few animals since the method allows amplification of the recovered target sites. It should allow the rapid characterization of the vast array of transcription factors involved in mental health and facilitate identification of therapeutically relevant targets altered by disease or drug abuse. Additionally, all of the reagents used will be obtained from companies within the USA, and the resource will be made available to the community through American depositories.